Zonula occludens toxin (“zot”) is an enterotoxin produced by Vibrio cholerae. Zot increases permeability by reversibly affecting the structure of tight junctions as described in WO 1996/37196. The comparison of its sequence with a potential human analogue (“zonulin”) revealed an 8-amino acid shared motif (GXXXVGXG, SEQ ID NO 23), described by D1 PIERRO, et al. Zonula Occludens Toxin Structure—Function Analysis. J. Biol. Chem. 276(22): 19160-19165 (2001).
The octapeptide of the following sequence: Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO 1) has been described as a peptide antagonist of zonulin by Fasano in WO 0007609 and by WANG, et al. Human Zonulin, a Potential Modulator of Intestinal Tight Junctions. Journal of Cell Science. 2000, 113:4435-4440 (2000). Fasano discloses its application in methods for treatment of gastrointestinal inflammation as well as of conditions associated with breakdown of the blood brain barrier. Fasano et al. also describe in U.S. Pat. No. 7,026,294 its use in a method for delay of onset of diabetes. Fasano describes delivery of the peptide to, inter alia, the small intestine via peptide-coated beads having a gastroresistant coating.
This octapeptide (“Larazotide”) is a promising treatment of various diseases that involve disordered intercellular communication, which include developmental and intestinal disorders leading to autoimmune disease (coeliac disease and type 1 diabetes), tissue inflammation, malignant transformation, and metastasis. None of the above references describe any process for the synthesis of this octapeptide. The present invention now makes available a process for its manufacture. The present invention also makes available various salts of the peptide suitable for pharmaceutical use. The present invention further provides solid state forms of such salts of the peptide and pharmaceutical compositions comprising the same. In one embodiment of the present invention, the salt of the peptide is crystalline (or highly organized). In a second embodiment, the crystalline form of the salt of the peptide may further comprise (stoichiometric and non-stoichiometric) waters of hydration (a “hydrate”). The present invention additionally provides pharmaceutical compositions comprising a crystalline stoichiometric or non-stoichiometric hydrate of a salt of the peptide. It is well recognized that pharmaceutical solids can exist in more than one solid state form (i.e., crystalline, noncrystalline/amorphous, quasicrystalline/organized aggregate). Polymorphism is defined as the ability of a solid compound to exist in more than one crystalline form with the same covalent chemical structure, but different supra-molecular structures and ordered arrangements of molecules within the crystalline lattice. In addition to exhibiting polymorphism, many pharmaceutical solids form hydrates and organic solvates, which themselves can be crystalline and exhibit polymorphism. Hydrates can be stoichiometric or non-stoichiometric. In a stoichiometric hydrate the water molecules are tightly associated with or bound to the pharmaceutical compound as well as to other water molecules and as a result are integral to the crystal lattice. In contrast, the water molecules of a non-stoichiometric hydrate (sometimes referred to as a variable hydrate) are loosely associated with the pharmaceutical compound and the crystal lattice. For example, in certain systems the water molecules of a variable hydrate reside in channels in the crystal lattice (Vogt et al. J Pharm Biomed Anal 40 (2006) 1080-1088). Generally speaking, the amount of water present in a non-stoichiometric or variable hydrate can be typically a function of the relative humidity (RH) environment of the sample. Hydrates, in particular non-stoichiometric hydrates, can be a difficult prospect for development because of the additional effort needed to investigate and characterize the different hydration states of the pharmaceutical compound. At the same time, the manufacturing process has to be scrutinized to determine what conditions are needed to ensure that the API contains a predictable composition.
It is well recognized that different solid state forms of the same compound can exhibit significantly different chemical and physical properties including color, morphology, stability, solubility, dissolution and bioavailability. As with all pharmaceutical compounds and compositions, the chemical and physical properties of a particular solid state form of a compound are important to its commercial development. These properties include, but are not limited to: (1) packing properties such as molar volume, density and hygroscopicity; (2) thermodynamic properties such as melting temperature, vapor pressure and solubility; (3) kinetic properties such as dissolution rate and stability (including stability at ambient conditions, especially to moisture, and under storage conditions); (4) surface properties such as surface area, wettability, interfacial tension and shape; (5) mechanical properties such as hardness, tensile strength, compactability, handling, flow and blend; and (6) filtration properties. These properties can affect, for example, processing and storage of pharmaceutical compositions, sometimes referred to as drug product and/or of the active pharmaceutical ingredient (API), sometimes referred to as drug substance. As mentioned above, different solid state forms of the API can have different rates of solubility which can translate into differences in bioavailability in vivo.
In general, the solid state form of a compound can be distinguished from another solid state form of the same compound by one or more of the following techniques: x-ray powder diffraction (XRPD), thermal techniques including thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), as well as Infrared (IR), Raman and/or solid state NMR (ssNMR) spectroscopy.
The Applicant describes here a process for the synthesis of the octapeptide Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO 1), which allows for an efficient production of said octapeptide with a good yield and a high quality and purity level while presenting advantages in terms of productivity and required manufacturing equipment.